Identification of Functional Domains in Herpes Simplex Virus 2 Glycoprotein B

Abstract

Glycoprotein B (gB) is one of four membrane proteins that are essential for the entry of herpes simplex viruses (HSV) into cells, and coexpression of the same combination of proteins in transfected cells results in cell fusion. The latter effect is reminiscent of the ability of virus infection to cause cell fusion, particularly since the degree of fusion is greatly increased by syncytial mutations in gB. Despite intensive efforts with the gB homologs of HSV and some other herpesviruses, information about functionally important regions in the 700-amino-acid ectodomain of this protein is very limited at present. This is largely due to the misfolding of the majority of the mutants examined. It was shown previously that the percentage of correctly folded mutants could be increased by targeting only predicted loop regions (i.e., not alpha-helix or beta-strand), and by using this approach new functional domains in HSV-2 gB have now been identified.Glycoprotein B (gB) is conserved throughout the Herpesviridae family and seems likely to have a function in membrane fusion for each virus, although significant details may vary and some homologs may have additional functions such as attachment. The gB homologs of herpes simplex virus 1 (HSV-1) and HSV-2, which share an 87% sequence identity, are essential for entry of the viruses into cells and for cell-to-cell spread (4,6,8,19,33). Nevertheless, much remains to be learned about regions of these proteins that are important for their functional activity. One approach to this problem was to characterize HSV-1 mutants resistant to neutralization by complement-independent anti-gB monoclonal antibodies (MAbs). This identified a small number of altered residues within the corresponding epitopes (14, 17), which are likely to be in a functionally important region of the protein. The mutations fall within a region comprising amino acids 273 to 298 (where the signal peptide is not included in the numbering), corresponding to amino acids 276 to 301 of HSV-2 gB. Another approach was targeted substitution of residues in the region adjacent to the transmembrane anchor; of those which could be mutated without disrupting protein folding, two (G716 and G736, equivalent to G721 and G741 of HSV-2 gB, excluding the signal sequence) were found to be important for virus entry (40). For some other HSV glycoproteins, such as gC, gD, and gH, functional domains have been mapped by linker-insertion mutagenesis, in which short oligonucleotides are ligated into restriction sites within the gene (7,12,15,35). This method has had only limited success with gB due to misfolding of the majority of the mutants (5,24,29).Combining experimental data with a secondary structure prediction made by the PHD neural network method (32), based on a multiple sequence alignment of 19 alphaherpesvirus gB sequences, it was shown previously that no mutants with insertions in predicted ␣-helices or ␤-strands folded correctly (24). In contrast, 50% of mutants with insertions in predicted loops, rather than ␣-helices or ␤-str...

Random linker-insertion mutagenesis to identify functional domains of herpes simplex virus type 1 glycoprotein B

“…Previously it was shown that certain 2-aa insertions into HSV-1 gB or the closely related HSV-2 gB (85% identity) also permitted cell surface expression (15,16). These insertions map to the N-terminal region outside the solved structure (S48, A104: insertion sites in HSV-2 gB at positions equivalent to HSV-1 gB T53 and T109) and to the small N-terminal segment of domain III (P130), linker 1 (R136), domain I (R189, Y254, R304, T313, P358), domain II (G381, S403, G437), the disordered region between domain II and linker 2 (P483), domain V (D680), and the cytoplasmic tail (E816).…”

“…Insertions after K70, K76, P80, and P81 had no effect on expression or function of gB, as assessed here. Similarly, insertions into HSV-2 gB at positions equivalent to HSV-1 gB T53 and T109 (S48 and A104 in HSV-2) had no effect on expression and only the insertion at A104 partially reduced function (16). Insertions K70 and K76 targeted a Lys-rich region from K68 to K76 shown to be critical for the binding of gB to heparin (20).…”

“…This loop was cleaved by trypsin in preparing the recombinant protein for crystallization and is a poorly conserved region where posttranslational cleavage occurs for members of the gB family encoded by other herpesviruses. Nine 2-amino acid insertions into HSV-1 or HSV-2 gB have been described to permit cell surface expression and at least partial function in cell fusion or viral entry (15,16). With the exception of one in the cytoplasmic tail and one in the disordered central loop, all map to the surface of the crystallized portion of gB, in domain I (Y254, R304, T313), domain II (G381, G437), the N-terminal-most segment of domain III (P130) and linker 1 (R136).…”

“…Overall, the crystal structure of gB reveals five distinct structural domains (I, II, III, IV, and V) and two linker regions in each monomer of the trimeric ectodomain. The apparent sequential roles of gH-gL and gB in HSV-induced membrane fusion and properties of each protein suggest a previously undescribed paradigm for fusion mediated by the herpesviruses.Previous mutagenesis studies of HSV gB to identify functional domains have had limited success because of protein misfolding and aberrant processing of most mutants (15)(16)(17). The fraction of mutants that were expressed on the cell surface and incorporated into virions could be increased by targeting mutations to regions predicted to lack secondary structure but, as a result, most mutants retained significant function or were misfolded despite the targeting.…”

“…Previous mutagenesis studies of HSV gB to identify functional domains have had limited success because of protein misfolding and aberrant processing of most mutants (15)(16)(17). The fraction of mutants that were expressed on the cell surface and incorporated into virions could be increased by targeting mutations to regions predicted to lack secondary structure but, as a result, most mutants retained significant function or were misfolded despite the targeting.…”